This project addresses one fundamental biological question that still remains poorly understood: What is the molecular substrate of hormonal regulation in response to chronic psychological stress? Understanding this question will not only lead us into deep insight of stress and adaptation, but it will also provide potential novel drug targets for the treatment of many stress-related disorders, such as major depression and anxiety. We will address this question by using a combination of functional genomic technology and classic methods of neuroanatomy and behavioral neuroendocrinology. In Specific Aim 1, we will use a combination of retrograde tract tracing and fluorescent active cell sorting (FACS) to purify the hypothalamic neurosecretory motor neurons (HNMN), and compare their global dynamic transcriptional profiles in response to four experimental conditions: control, acute stress, chronic stress, and delayed response, in order to identify the chronic-stress-vulnerable genes. In Specific Aim 2, we will use combined genetic labeling and retrograde tract tracing methods to double-label a subpopulation of the HNMN, the CRH neuroendocrine neurons;and we will also use FACS to isolate this population and extract its RNA. Then, we will use the more sensitive and precise quantitative RT-PCR method to screen 24 candidate genes with these CRH-specific RNA samples. These candidate genes will be pre-selected from (1) the candidate gene pool generated in Specific Aim 1 and (2) the online gene expression database-the Allen Brain Atlas (ABA). Combining these two Specific Aims, we will pinpoint the cell-type specific genes involved in the regulation CRH transcription during chronic psychological stress, which will be used immediately for our future research to explore the genetic etiology of chronic stress. We will upload all data into a public accessible online database system of GEO and/or ArrayExpress. This proposal provides a novel strategy for applying functional genomics to address deep biological questions in the classic fields of behavioral neuroendocrinology, and is conceptually and technically demanding across multiple disciplines. If successful, this work could significantly advance our understanding of stress physiology and yield key insights into the endocrine response to stress associated with a number of neuropsychiatric disorders. Thus, this proposal fits into the R21 mechanism very well, which is meant for high risk but highly innovative and potentially high impact projects. The potential for a more profound understanding of the stress response, both acute and chronic, make this a very compelling proposal. To ensure the success of this project, we have established expertise in our laboratory and have developed a strong collaboration with several other local laboratories with exceptional expertise in functional genomics, genetics, and behavioral neuroendocrinology. We have also performed extensive preliminary studies and proven that all experimental procedures proposed here are feasible and reliable. PUBLIC HEALTH RELEVANCE Chronic psychological stress and mood disorders are highly prevalent within our population, and are associated with high levels of morbidity and mortality, as well as great economic cost. The lack of knowledge of the underlying molecular pathogenesis of psychological stress has prevented the exploration of more effective approaches of prevention and treatment. This proposal is a pilot project of our long-term objective that aims to determine the neural circuits and molecular mechanisms underlying chronic psychological stress. We will apply state-of-the-art FACS-array technology to determine molecular differences between acute and chronic stress, and identify hormonal regulation genes that are vulnerable to chronic psychological stress. These genes will provide candidates for exploring the precise genetic etiology of chronic stress and developing novel drug targets. Thus, this study has great value clinically and for public health.